Angiogenesis, the process of new blood vessels formation, is a complex process involving the coordinated interaction of numerous cell types. The critical cells are the endothelial cells, which contain all of the genetic information necessary to form primitive tubes and branches. Other cells, such as smooth muscle cells, mast cells, and macrophages release important modulators of angiogenesis. Hypoxia, decreased blood flow, and released angiogenic substances such as vascular endothelial growth factor (VEGF) can trigger angiogenesis. The process begins with a breakdown of the extracellular matrix, followed by proliferation and migration of endothelial cells into the tissue. Initially the endothelial cells form cords. Later large vacuoles form in the cells, leading to the formation of tubes. The endothelial tubes have a lumen, but are abnormally permeable and leaky until pericytes are recruited to reinforce the new vessels. Several growth factors, most notably VEGF, bFGF, and angiopoetin-1, promote angiogenesis. VEGF, a specific mitogen for endothelial cells, can independently stimulate new vessel growth. However, overexpression of VEGF in developing avian embryos results in large vessels that are leaky, which leads to tissue edema. The coordinated effects of several growth factors may be necessary to stimulate the development of normal new vessels. Hence, finding ways to use upstream modulators in a tissue-specific way may provide a therapeutic advantage over the application of individual growth factors.
VEGF is a direct, or primary, angiogenic factor, meaning that it is able by itself to induce angiogenesis in endothelial cells in vitro or in vivo. Secondary, or indirect, angiogenic factors work by causing cells to release primary factors. Experts fear that using primary factors clinically will cause pathologic angiogenesis in other tissues. Thus, a limitation of using adenosine or other promoters of angiogenesis could be new vessel growth in healthy as well as diseased tissues. Hence, activation of upstream secondary angiogenic stimuli may produce more regulated and normal vascular growth. Additionally, the ability to target angiogenic stimulation to specific tissues would diminish the risk of indiscriminate angiogenesis.
There are widespread clinical applications for the stimulation of the angiogenesis in cardiovascular medicine and ophthalmology. Stimulating new vasculature in ischemic tissues, especially heart and limbs is currently an active clinical endeavor because it could have a major impact on morbidity and mortality from atherosclerosis. Trials in humans have shown the usefulness of VEGF in stimulating collateral vessels to ischemic lower extremities, improving ulcer healing and decreasing limb loss. There are also ongoing clinical trials using VEGF infusions in patients with intractable, inoperable angina pectoris.
Abundant evidence shows that hypoxic or ischemic tissues release adenosine and that adenosine stimulates angiogenesis. Possible mechanisms of vessel growth include increased flow, stimulation of vascular cell proliferation and migration, or stimulation of growth factor secretion. Some of the results obtained in previous studies on adenosine effects in vivo and in vitro have suggested that activation of adenosine A2 receptors (A2A or A2B) are responsible for the ability of adenosine to stimulate angiogenesis. The activation of A2B receptors on cultured endothelial cells has been shown to stimulate VEGF release, but A1 adenosine receptor activation seemed to play little or no role. The present invention, however, demonstrates the A1 receptor is more important than has been previously thought. Indeed, the present invention relates to the use of thiophene derivatives as allosteric enhancers of agonist activity at adenosine A1 receptors. Allosteric enhancers of A1 adenosine receptors selectively stimulate angiogenesis in ischemic tissue and not in tissue that has adequate blood flow. This site-specificity represents a major advantage over other angiogeneic agents that are not selective for ischemic tissue.
Adenosine triggers endothelial cell proliferation in cultured cells and angiogenesis in animal models. Adenosine is a logical modulator for the hypoxic stimulation of angiogenesis. It is a metabolite of ATP released from all ischemic or hypoxic tissues, where it acts as a “retaliatory metabolite” to restore normal oxygen delivery, initially by dilating existing blood vessels. Chronic hypoxia has long been considered a driving force for new blood vessel formation. Increased vascular density is seen in humans at high altitudes, in chronically stimulated skeletal muscle, and in rapidly growing tumors. Hypoxia initiates proliferation of cultured endothelial cells that can be blocked by unselective adenosine receptor antagonists. Subtype-selective ligands have been used to tease out the mechanism of adenosine-induced endothelial cell proliferation and migration, but the results have been inconsistent. The chicken chorioallantoic membrane (CAM) model is a suitable vehicle for studying the effect of adenosine on angiogenesis. In this model lowering oxygen concentration stimulates neovascularization, but adenosine has not been consistently angiogenic. Receptor subtype-selective ligands have not previously been tested in the CAM.
Adenosine acts via four types of cell surface, G protein-coupled receptors, A1, A2A, A2B and A3. A1 and A3 receptors are the most similar in amino acid sequence and pharmacology. These receptors couple to G proteins from the Gi/Go family and inhibit adenylyl cyclase. Stimulation of A1 and A3 receptors can also activate phospholipase C, presumably via G protein sub-units. A2A and A2B receptors couple to Gs and stimulate adenylyl cyclase, but the A2B receptor can also couple to Gq. In the heart, A1 receptors have negative chronotropic, dromotropic and inotropic effects. The A1 receptor, and perhaps the A3, is also involved in the preconditioning phenomenon, which protects ischemic tissues. Coronary arteries express A2A receptors; their activation results in coronary vasodilation. A2A receptors also occur on leukocytes, where they attenuate the inflammatory response and thereby decrease reperfusion injury. Accordingly, adenosine acts in a number of ways to protect ischemic tissues; it decreases metabolism, increases blood flow, and attenuates inflammatory injury. Adenosine activates A2B receptors on cultured endothelial cells to trigger VEGF release and endothelial mitogenesis. Adenosine also appears to stimulate angiogenesis, but to date no attempt has been made to define the adenosine receptor subtypes involved in the CAM model. Additionally, heretofore, it had not been shown that adenosine stimulates angiogenesis in adult mammalian models. The development of more selective adenosine receptor ligands and cloning of the chicken A1, A2A, and A3 receptors have enabled us to identify adenosine receptor subtypes participating in the angiogenic response of CAM.
Allosteric enhancers of receptors are defined as compounds that bind to an allosteric site distinct from the binding site of the endogenous ligand and potentiate responses to agonists. Benzodiazepine anxiolytics and calcium channel blockers are familiar examples of drugs that act allosterically. Allosteric enhancers of adenosine A1 receptors act only on the adenosine-receptor-G protein ternary complex. Accordingly, they have little effect by themselves, but enhance the actions initiated by A1 receptors when increases in endogenous adenosine levels in ischemic tissues increase receptor occupancy.
PD 81,723 (PD) is the archetype of a family of arninothiophenes that were the first described allosteric enhancers of adenosine A1 receptors. These compounds increase binding of [3H]N6-cyclohexyladenosine (CHA) to adenosine A1 receptors and caused a functional enhancement of the effects of adenosine A1 receptor activation in various tissues. PD is selective for adenosine A1 receptors, having no effects on other adenosine receptor subtypes or on other classes of receptors. PD has shown enhancement at A1 receptors of all species tested to date. In the absence of adenosine or A1-selective agonists, the enhancer molecules alone act as very weak antagonists for adenosine receptors. Despite PD demonstrating allosteric enhancer activity, there still remains a need for compounds having improved allosteric enhancer activity.
The administration a compound that promotes angiogenesis can be an effective method for treating stroke, heart disease, peripheral vascular disease. The administration of such compound can also be an effective method for treating cardiac arrhythmias, chronic pain and inducing sleep. The ability of the improved allosteric enhancers described herein to promote angiogenesis in two animal model systems, the chicken chorioallantoic membrane model and the rat mesenteric model, demonstrates that allosteric enhancers of the adenosine A1 receptor enhance the ability of adenosine to promote new vessel growth.
Accordingly, the present invention provides novel thiophene derivatives to be used as improved agonist allosteric enhancers at adenosine A1 receptor.
Additionally, the present invention provides an original therapeutic method for preventing or treating a pathological condition or symptom in a mammalian subject, such as a human, wherein increased angiogenesis is desired, by administering to a mammal in need of such therapy an effective amount of the aforementioned adenosine A1 receptor allosteric enhancer.